Identification of RIPK3 as a target of flavonoids for anti-necroptosis in vitro

Receptor-interacting protein kinase 3 (RIPK3), a key regulator of necroptosis, has emerged as an important target for therapeutic intervention. Flavonoids are natural compounds known for their anti-inflammatory and antioxidant properties, with recent studies highlighting their potential to modulate necroptosis. In this study, we explored the potential of RIPK3 as a target for flavonoids to achieve anti-necroptosis and anti-inflammatory effects. A library of 63 flavonoids was tested for RIPK3 binding and kinase inhibition using fluorescence polarization (FP) competition assay and ADP-Glo kinase activity assay. Six flavonoids, including scutellarein, robinetin, baicalin, myricetin, baicalein, and tricetin, showed significant inhibition of RIPK3, with IC₅₀ values ranging from 2.5 to 13.7 μM. Structural studies of tricetin and robinetin through co-crystallization and molecular docking revealed distinct binding modes of these flavonoids within the ATP-binding pocket of RIPK3. The anti-necroptosis effects of these flavonoids were further evaluated in human HT-29 cells and mouse embryonic fibroblasts (MEFs) using a TSZ-induced cell death assay, resulting in EC₅₀ values in the tens of micromolar range. Western blot analysis demonstrated that these flavonoids inhibit the phosphorylation of RIPK3 and its downstream effector, mixed lineage kinase domain-like protein (MLKL), and disrupt the formation of RIPK1 and RIPK3 aggregates in the necroptosis pathway. These findings identify RIPK3 as a target of natural flavonoids for the first time and elucidate the molecular mechanism underlying the anti-necroptotic activity of these flavonoids.